Determination of lipid levels in a body fluid, particularly determination of total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides, apolipoprotein, phospholipids, sphingolipids and cholesteryl esters levels, collectively referred to as “lipids”, has rapidly come into wide use with the recent development of enzymatic, immunologic, electrophoretic and ultracentrifugation determination processes. As a result, the utility of these lipids in the field of clinical diagnosis has been increasing. Therefore, a proper standard solution for determination of lipid levels is required.
Most cholesterol analyses comprise mixing a chromogenic working reagent with the sample to be analyzed and, after color development, observing color intensity on a spectrophotometer. The working reagent generally contains cholesterol esterase in order to convert cholesterol esters to free cholesterol, and cholesterol oxidase to oxidize free cholesterol yielding cholestenone, simultaneously liberating hydrogen peroxide. The amount of cholesterol present is then determined by measuring the amount of hydrogen peroxide liberated using the peroxidase/phenol/4-aminoantipyrine system, also present in the working reagent. This analysis is also run on calibrator solutions, which are prepared by adding the working reagent to standard solutions containing known concentrations of cholesterol, so that meaningful quantitative information can be obtained.
Most HDL Cholesterol analyses are performed by homogeneous assays without the need for pretreatment of centrifugation steps. The method depends on a unique detergent which solubilizes only the HDL lipoprotein particles and releases HDL cholesterol to react with the enzymatic assay described above to produce a color product. The same detergent also inhibits the reaction of the cholesterol enzymes with LDL, very-low-density lipoproteins (VLDL) and chylomicrons lipoproteins by adsorbing to their surfaces. Furthermore, a polyanion contained in the reagent enhances the selectivity for HDL cholesterol by complexing the LDL, VLDL and chylomicron proteins (see Beckman Synchron CX Systems Chemistry Information Manual No. 249595, May 2000).
HDL Cholesterol analyses are also measured via a direct method using polyethylene glycol (PEG)-modified enzymes and dextran sulfate. When cholesterol esterase and cholesterol oxidase enzymes are modified by PEG, they demonstrate selective catalytic activities toward lipoprotein fractions, with the reactivity increasing in the order: LDL<VLDL<chylomicrons<HDL. In the presence of magnesium ions, sulfated α-cyclodextrin reduces the reactivity of cholesterol, especially in chylomicrons and VLDL, without the need for precipitation of lipoprotein aggregates. (See Roche Diagnostics Corporation, GmbH, 2002, No. 05453101, Mannheim, Germany.) This allows for the selective determination of HDL cholesterol in serum.
To date, LDL Cholesterol assays have been performed taking advantage of the selected micellary solubilization of LDL-cholesterol by a nonionic detergent and the interaction of a sugar compound with lipoproteins (VLDL and chylomicrons). When a detergent is included in the enzymatic method for cholesterol determination, the relative reactivities of cholesterol in the lipoprotein fractions increases in this order: HDL<chylomicrons<VLDL<LDL. In the presence of magnesium cations, a sugar compound markedly reduces the enzymatic reaction for the cholesterol measurement in VLDL and chylomicrons. The combination of a sugar compound with detergent enables the selective determination of LDL cholesterol in serum. (See Roche Diagnostics Corporation, GmbH, 2002, Technical Publication No. 054565801, Mannheim, Germany). LDL cholesterol is also determined using procedures described in technical bulletins available from, among others, Genzyme Corporation, Beckman Coulter, and the like.
Most triglycerides assays comprise mixing a chromogenic working reagent with the sample to be analyzed and, after color development, observing color intensity on a spectrophotometer. The working reagent generally contains lipase to convert triglycerides to glycerol and fatty acids, followed by the reaction of glycerol and ATP catalyzed by glycerol kinase to produce glycerol-3-phosphate and ADP. The glycerol-3-phosphate is then catalyzed with glycerophosphate oxidase to form dihydroxyacetone phosphate plus hydrogen peroxide. The amount of triglycerides present is then determined by measuring the amount of color from the reaction of peroxidase converting chromogenic substances such as 4-aminophenazone and 4-chlorophenol or 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid to form the end product for photometric measurement. This analysis is also run on calibrator solutions, which are prepared by adding the working reagent to standard solutions containing known concentrations of triglycerides, so that meaningful quantitative information can be obtained.
Apolipoprotein A-1 (APO-A1) is measured using immuno-turbidimetric assay techniques wherein sample is added to a Tris buffer followed by addition of an anti-lipoprotein A-1 antibody. Anti-lipoprotein A-1 antibodies react with the antigen in the sample to form antigen/antibody complexes which, following agglutination, are measured turbidimetrically. (See Roche Diagnostics Corporation, GmbH, 2002, Technical Publication No. 03032612, Mannheim, Germany).
Apolipoprotein B (APO B) is often measured using immuno-turbidimetric assay techniques wherein sample is added to a Tris buffer followed by addition of an anti-lipoprotein B antibody. Anti-lipoprotein B antibodies react with the antigen in the sample to form antigen/antibody complexes which, following agglutination, are measured turbidimetrically. (See Roche Diagnostics Corporation, GmbH, 2002, Technical Publication No. 03032639, Mannheim, Germany).
These procedures, whether enzymatic, immunologic or turbidimetric, all require that the working reagent, calibrators, quality control (QC) materials and samples be of an aqueous matrix, such as, but not limited to, serum, plasma, and the like. The need for this specific matrix presents a problem with respect to the preparation of calibrator solutions, since cholesterol, and most other components commonly referred to as “lipids”, are substantially insoluble in water. For example, a cholesterol standard solution made in alcohol, which when combined with the aqueous working reagents, can produce precipitation of cholesterol. This yields an unacceptable calibrator solution. Such precipitation can lead to uncertain or incompatible color development in the calibrator, QC or calibration verification and/or linearity materials and generally makes the selected specimen useless for reliably correlating color intensity with cholesterol concentration. In a second example, measurement of triglycerides with a reference method uses a triglycerides standard made up of triolein and/or tripalmitin in an alcohol matrix.
Due to their hydrophobicity, lipids are generally dissolved in an organic solvent. These solutions can then be used in the Abell-Kendall reference method for cholesterol determination. In this technique, the solvent solubilized cholesterol is compatible with the assay methodology. However, the lipids in these standard solutions differ in their existing state and fluid property from those found in serum. Thereby, the reactivity between the standard solution and the body fluid (e.g., serum) differ, resulting in errors in the determined values.
Therefore, human- or animal-derived serum or purified lipoprotein cholesterol has been used as the standard solution. Triglycerides may alternately be calibrated using materials such as an artificial water-soluble glycerol, water insoluble animal egg yolk extracts, endogenous triglycerides or water-insoluble solvent-based (alcohol) triolein/tripalmitin standards. For triglyceride calibration, the use of glycerol is not preferred in cases where free glycerol may be present in the sample thus necessitating a “glycerol blanking” prior to lipase conversion of the triglycerides to glycerol in the reaction scheme. The National Institute for Standards and Technology (NIST) has Standard Reference Materials (SRM) available for Total Cholesterol, Total Glycerides, Triglycerides, HDL-Cholesterol and LDL-Cholesterol. These SRMs (SRM 1951a and 1951b) consist of reference concentration values that are lot-specific. The matrixes for these SRMs consists of “Lipids in Frozen Human Serum”, shipped under dry ice and are stable for only one (1) week at −20° C. and at −80° C. for longer periods of time. These media all present high costs, difficult shipping conditions, requirements for a −80° C. storage freezer upon receipt, can contain unknown impurities that can influence the determined values, and present technical difficulties relating the potential presence of infectious agents. Additionally, processes for purifying lipoproteins are technically complex and costly to perform for routine analyses of vast numbers of samples.
Consequently, lipids and/or lipoproteins and other constituents of interest, have been solubilized in water containing a surfactant and used as the standard/reference solution. However, a large amount of surfactant is required for the solubilization of lipids in water. This, in turn, increases the viscosity of the solution making its handling sometimes difficult. Moreover, samples comprising of solubilized lipids, and/or lipoproteins are unstable and demonstrate short shelf life upon storage. To combat this issue, solubilized lipid solutions have been lyophilized. However, post reconstitution of these samples, there is subsequent loss of constituent activity.
Despite the difficulties in preparing known standards for use in various assays, in order to convert a detectable assay signal to a quantitative result (e.g., concentration of the constituent), the assay must first be calibrated. Calibration is performed by running the assay first with a series of samples of predetermined concentrations following instructions from the manufacturer of the assay method used. These samples are referred to as “calibrators.” The results obtained by reading the signals from the calibrators are used to generate, by using various curve fit models, a calibration curve spanning the entire assay range. The calibration curve can then be used to determine the concentration of an unknown sample of QC, calibration verification/linearity, or unknown sample from the signal it produces. Since the calibrator often responds differently than patient serum or other test sample, changes in the calibrator with time may render it ineffective or cause erroneous or inaccurate test results. In the art, there is a need for more stable aqueous calibrators and calibration curves, as well as for quality control and calibration verification/linearity materials to assess the continued accuracy of an assay. The present invention meets these needs.